The dlg tumor suppressor gene of Drosophila gives rise to a protein, DlgA, that is localized at septate junctions between epithelial cells. Mutations in this gene lead to neoplastic overgrowth of the imaginal discs in the larva, followed by death during the pupal stage. DlgA is the prototype for the MAGUK family of proteins that are the subject of this Program Project. Our goal is to understand the role of DlgA in septate junction structure and in cell proliferation control. The domain(s) responsible for localization of DlgA to the septate junction will be identified by examining the localization of epitope-tagged parts of the protein produced in vivo from expression constructs. The proteins that bind to DlgA will be identified by affinity purification or co- immunoprecipitation, followed by tests with specific antibodies to candidate binding proteins, or by microsequencing of purified binding proteins. The yeast two-hybrid system will also be used to search for DlgA-interacting proteins. Candidate proteins will be tested directly for DlgA binding, and any binding domains will be mapped in collaboration with Dr. Chishti. The genes encoding DlgA-interacting proteins will be cloned and the structure of the proteins predicted from cDNA sequence. We will also use a genetic approach in Drosophila to select modifiers of dlg that may identify interacting proteins. In collaboration with Dr. Chishti, the GUK domain of DlgA will be tested for guanylate kinase and GMP-binding activities, and in collaboration with Dr. Anderson, the DlgA protein will be tested for tyrosine phosphorylation. A newly identified Drosophila gene encodes a protein, CAMGUK, that shows homology to MAGUKs but also contains an N-terminal extension with highly significant similarity to calcium/calmodulin-dependent protein kinase (CAM kinase). The CAM kinase as well as the GUK domains of CAMGUK will be produced in bacteria or animal cells and tested for their respective catalytic activities, and the functions of other CAMGUK domains will be assayed using the same methods as for DlgA. Genetic methods will be used to determine the function of CAMGUK in the cell and during development. We also propose to begin a molecular genetic analysis of a recently identified additional Drosophila MAGUK, Dlg-2, that shows strong homology to human Hdlg. The work will help to elucidate the functions of domains in DlgA and CAMGUK as well as in MAGUKs in general.